Drosophila polo kinase is required for cytokinesis - PubMed (original) (raw)
Drosophila polo kinase is required for cytokinesis
M Carmena et al. J Cell Biol. 1998.
Abstract
A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.
Figures
Figure 1
A giant gonial cell resulting from failure of pre-meiotic cytokinesis in testes from homozygous polo1 males. Staining reveals microtubules (left), γ-tubulin distribution revealing centrosomes (middle), and chromatin (right). Multiple monopolar spindles originate from as many γ-tubulin–containing foci, suggesting that centrosome duplication and separation continue in the absence of cytokinesis.
Figure 9
Transmission electron micrographs of polo1 (A, C, E) and wild-type (B, D, F) cysts of spermatids at sequential stages of elongation. (A) Cross section of an early elongating polo1 spermatid showing three axonemes (large arrowheads) associated with only one mitochondrial derivative (small arrowhead). The structure of one axoneme appears abnormal (large arrowhead). A fourth axoneme is outside the frame of the picture. (B) In contrast, in wild-type early elongating spermatids one axoneme (large arrowhead) is normally associated with a pair of mitochondrial derivatives (small arrowheads). (C) Cross section of elongating cysts at a later stage shows that spermatids in polo1 males may contain four axonemes (cell indicated by arrowhead). (D) Wild-type spermatids at a similar stage contain single axonemes. (E) Section of mature sperm from polo1 males showing irregular numbers of axonemes (two in the cell indicated by the large arrowhead, three in the cell indicated by the small arrowhead). (F) Mature wild-type sperm contain single axonemes. Although the occasional abnormal axoneme is seen in the polo1 mutant (A), the majority of axonemes from polo1 and wild-type males have an identical structure throughout the sequential stages of elongation: they comprise one pair of central tubules surrounded by nine doublets, and nine accessory tubules in later stages.
Figure 2
Cytokinesis defects in the pre-meiotic divisions are indicated by reduced numbers of ring canals, and enlarged cells in cysts of primary spermatocytes. Optical sections of a cyst of (A) wild-type and (B) polo1 mutant primary spermatocytes. DNA is stained blue, microtubules in green and Pav-KLP in red. The wild-type cyst contains 16 cells inter-connected through 15 ring canals that contain Pav-KLP. The polo1 mutant cyst contains 13 cells connected by 12 ring canals. One cell (arrow) is considerably larger than its neighbors. Bars, 20 μm.
Figure 3
polo1 spermatocytes show spindle mid-zone defects at late anaphase. Progression through the first meiotic division in polo1 is shown in the main panels, and in wild-type in the insets. Testes preparations have been stained with antibodies against β-tubulin (left-hand panels), γ-tubulin (middle panels), and Hoechst to reveal DNA (right-hand panels) (see Materials and Methods). (A) Mature spermatocytes. Note the orthogonal rod-like γ-tubulin structures at the focus of the cytoplasmic microtubules. (B) Metaphase spermatocytes. γ-Tubulin is transformed into more diffuse bodies at the poles of the spindles. Bivalents congregate in single masses. (C) Late anaphase spermatocytes. A prominent mid-zone structure strongly stained for β-tubulin (arrowhead in insets) is present in wild-type anaphase spindles, whereas no such structure is found in polo1 spindles (arrows). γ-Tubulin is present in this region in both mutant and wild-type. (D) Telophase spermatocytes. Wild-type spermatocytes show characteristic mid-bodies with a distinct gap in β-tubulin staining between the two halves of the spindles (arrowheads in insets); in contrast the spindle microtubules of polo1 mutant spermatocytes fail to organize similar structures during telophase (arrows). Whereas in the wild-type, the γ-tubulin in the central region of the spindle begins to redistribute towards the poles, in the mutant cell it remains in the central region.
Figure 4
Tetrapolar cells in which cytokinesis has failed in both meiotic divisions in the polo1 mutant. Testes have been stained to reveal microtubules (left), γ-tubulin (middle), and chromatin (right). Each cell contains four astral microtubules and four foci of γ-tubulin (arrows). Chromosomes frequently appear to undergo non-disjunction (arrowheads).
Figure 5
Mislocalization of Pav-KLP and Peanut at telophase in polo1 mutants. In all of these panels, DNA is stained blue, and Pav-KLP red. The third immunostain (green) in C and D reveals microtubules, and in E and F, the septin Peanut. (A) A wild-type cyst at late anaphase/early telophase I in which Pav-KLP can be seen to be present in the ring canals (arrowhead) and the contractile rings (arrow). (B) In an equivalent polo1 mutant cyst, Pav-KLP (red) is localized in the contractile ring (arrow) and remains in the ring canal in normal cells. In spermatocytes showing the mutant phenotype, Pav-KLP accumulates in the polar regions (small arrowhead) and does not redistribute to the midbody. (C) A telophase I cyst showing the distribution of Pav-KLP (red) relative to microtubules (green). Large arrow, a cleavage ring; and small arrowhead, a ring canal. In addition, Pav-KLP is associated with some (putatively immature) axonemal microtubules (small arrow), but not with others (large arrowhead). (D) Telophase figures in polo1 in which Pav-KLP is associated with early (arrow) and late (small arrowhead) cleavage rings in cells undergoing apparently normal late meiotic events. Cleavage rings fail to form correctly in the multipolar figure (large arrow). (E) Immunolocalization of Pav-KLP (red) and Peanut (green) in a wild-type late anaphase/early telophase I cyst. The large arrow indicates an early cleavage ring in which the staining for Peanut is more pronounced than for Pav-KLP. The two proteins colocalize in the later cleavage ring (small arrow) with Pav-KLP being more concentrated towards the inner side. Ring canals appear to contain Pav-KLP and not Peanut (small arrow). (F) polo1 mutant cyst with Pav-KLP and Peanut colocalizing in both early (large arrow) and late cleavage rings. Punctate staining of Pav-KLP is also seen in the regions of the spindle poles (small arrows).
Figure 6
Abnormal actin ring formation during cytokinesis in polo1 mutants. DNA is stained blue; microtubules are stained green; and actin is stained red. Actin rings at equivalent stages can be seen in the telophase figures from the wild-type cyst. In the polo1 mutant cyst, actin forms a ring in late telophase cells with a normal spindle mid-body (small arrow), but not in cells in which the mid-body has not formed (large arrow). Ring-like structures of actin may (small arrowhead) or may not (large arrowhead) form in cells with tripolar spindles. Bars, 10 μm.
Figure 7
Cyclin B is degraded upon meiotic exit in polo1 mutants. Metaphase–telophase mutant figures in the polo1 mutant. This cyst has several multipolar cells at stages both before and after cyclin B degradation. The large and small arrows indicate cells with a tetrapolar and abnormal bipolar spindles respectively in which cyclin B has undergone degradation. Wild-type cyst with cells showing progressive cyclin B degradation at various stages of the metaphase-anaphase transition.
Figure 8
Multinuclear spermatids in polo1. (A and B) Squashed preparations of onion stage spermatids. The clear spherical structure is the nucleus, and the dark spherical structure the mitochondrial derivative or Nebenkern. A binucleate cell with a single Nebenkern is indicated by the arrowhead in A. C and D show larger cells that have 5 or 4 nuclei, respectively and a single Nebenkern. D shows spermatids at an early stage of elongation. The arrowhead points to a binucleate cell.
Figure 10
A schematic illustration of spermatogenesis in Drosophila illustrating some of the defects that arise as a result of the failure of cytokinesis. Color is used to depict DNA (blue); ring canals (red); the fusome (ochre), a structure rich in membranes and spectrin that passes through the ring canals; microtubules (green); and mitochondrial derivatives (black).
References
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